Recombinant glycoprotein vaccines are currently in development for several herpesviruses, including HSV and EBV, and for HIV. The EBV membrane antige (EBV-MA) gp350 is a glycoprotein which contains virus neutralizing epitopes and is currently being tested as a possible EBV vaccine candidate in England. A series of 14 monoclonal antibodies (mAbs) to EBV-MA were obtained from Louis Qualtiere and Gary Pearson. These have been used to screen antigen expressing clones for reactivity to different epitopes in order to identify the nucleic acid sequences important for virus neutralization. Initially ten overlapping clones which express different portions of unglycosylated gp350 were constructed in E. coli. Four of 14 mAbs tested reacted with the recombinant E. coli antigens, and three antigenic epitopes (nucleotides 1980-2307, 3186-3528 and 3528-3576 in Bam H L) identified. Proteins expressed by the clones were used to produce antisera in rabbits. When tested for neutralization of EBV in tissue culture, the rabbit antisera as well as the mAbs which recognized the three epitopes failed to neutralize, suggesting that these epitopes are not involved in neutralization. Since the remaining mAbs may only recognize the full-length glycosylated form of gp350, the entire gp350 coding sequenc has also been expressed in a Baculovirus expression system which produces glycosylated protein. All of the MAbs react with the full-length baculovirus-expressed protein. Immunofluorescence studies of Sf9 cells expressing gp350 suggest the protein is membrane associated. Five additional subclones which express different portions of gp350 in the Baculovirus expression system have been prepared and used for additional epitope mapping studies. Like the E. coli-expressed fragments these subgenomic proteins only react with 4 of the 14 mAbs which recognize the entire Baculovirus-expressed gp350. Deglycosylated gp350 retained reactivit with the mAbs, while denatured gp350 lost reactivity with most of the mAbs, suggesting that correct conformation rather than glycosylation is required for recognition. A manuscript describing these data is in press in J. Gen. Virol. Additional studies comparing the immunogenicity of native versus denatured baculovirus-expressed gp350 in rabbits are in progress.